Intragenic DNA Methylation Regulates Insect Gene Expression and Reproduction through the MBD/Tip60 Complex

DNA methylation is an important epigenetic modification. However, the regulations and functions of insect intragenic DNA methylation remain unknown. Here, we demonstrate that a regulatory mechanism involving intragenic DNA methylation controls ovarian and embryonic developmental processes in Bombyx mori. In B. mori, DNA methylation is found near the transcription start site (TSS) of ovarian genes. By promoter activity analysis, we observed that 5′ UTR methylation enhances gene expression. Moreover, methyl-DNA-binding domain protein 2/3 (MBD2/3) binds to the intragenic methyl-CpG fragment and recruits acetyltransferase Tip60 to promote histone H3K27 acetylation and gene expression. Additionally, genome-wide analyses showed that the peak of H3K27 acetylation appears near the TSS of methyl-modified genes, and DNA methylation is enriched in genes involved in protein synthesis in the B. mori ovary, with MBD2/3 knockdown resulting in decreased fecundity. These data uncover a mechanism of gene body methylation for regulating insect gene expression and reproduction

BmILF and I-motif Structure Are Involved in Transcriptional Regulation of \u3cem\u3eBmPOUM2\u3c/em\u3e in \u3cem\u3eBombyx mori\u3c/em\u3e

Guanine-rich and cytosine-rich DNA can form four-stranded DNA secondary structures called G-quadruplex (G4) and i-motif, respectively. These structures widely exist in genomes and play important roles in transcription, replication, translation and protection of telomeres. In this study, G4 and i-motif structures were identified in the promoter of the transcription factor gene BmPOUM2, which regulates the expression of the wing disc cuticle protein gene (BmWCP4) during metamorphosis. Disruption of the i-motif structure by base mutation, anti-sense oligonucleotides (ASOs) or inhibitory ligands resulted in significant decrease in the activity of the BmPOUM2 promoter. A novel i-motif binding protein (BmILF) was identified by pull-down experiment. BmILF specifically bound to the i-motif and activated the transcription of BmPOUM2. The promoter activity of BmPOUM2 was enhanced when BmILF was over-expressed and decreased when BmILF was knocked-down by RNA interference. This study for the first time demonstrated that BmILF and the i-motif structure participated in the regulation of gene transcription in insect metamorphosis and provides new insights into the molecular mechanism of the secondary structures in epigenetic regulation of gene transcription

Identification, Expression and Target Gene Analyses of MicroRNAs in Spodoptera litura

MicroRNAs (miRNAs) are small RNAs widely present in animals and plants and involved in post-transcriptional regulation of gene transcripts. In this study we identified and validated 58 miRNAs from an EST dataset of Spodoptera litura based on the computational and experimental analysis of sequence conservation and secondary structure of miRNA by comparing the miRNA sequences in the miRbase. RT-PCR was conducted to examine the expression of these miRNAs and stem-loop RT-PCR assay was performed to examine expression of 11 mature miRNAs (out of the 58 putative miRNA) that showed significant changes in different tissues and stages of the insect development. One hundred twenty eight possible target genes against the 11 miRNAs were predicted by using computational methods. Binding of one miRNA (sli-miR-928b) with the three possible target mRNAs was confirmed by Southern blotting, implying its possible function in regulation of the target genes

Protein profiles of the midgut of Spodoptera litura larvae at the sixth instar feeding stage by shotgun ESI-MS approach

By using shotgun HPLC-ESI-MS proteomics approach, 2043 peptides were identified from the midgut of Spodoptera litura larvae at the sixth instar feeding stage, out of which 1489 (72.9%) were found to have their homologues in the public protein databases and 842 had identities of molecular functions. Seven-hundred forty-one peptides were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization, with 336 and 251 peptides being related to catalytic activity and binding activity, respectively. Most of the catalytic proteins had activity of hydrolases, oxidoreductases and transferases and most of the binding proteins were involved in protein-binding activity. Among the annotated peptides, 487 were classified into different cellular processes and 490 were classified to locate in the cytoplasm. Nonredundant enzymes associated with the metabolisms of carbohydrates, lipids and fatty acids, amino acids and proteins, translation, transport, and stress resistance were identified. Presence and expression at high levels of numerous enzymes of glycolysis pathway, synthesis of proteins, and absorption and transport of fatty acids and lipids indicate that active metabolism processes of carbohydrates, proteins, and lipids occurred in the midgut of sixth instar feeding larvae of S. litura. The protein profile provides a basis for further study of the physiological events in the midgut of S. litura

A New Processing Chain for Real-Time Ground-Based SAR (RT-GBSAR) Deformation Monitoring

Due to the high temporal resolution (e.g., 10 s) required, and large data volumes (e.g., 360 images per hour) that result, there remain significant issues in processing continuous ground-based synthetic aperture radar (GBSAR) data. This includes the delay in creating displacement maps, the cost of computational memory, and the loss of temporal evolution in the simultaneous processing of all data together. In this paper, a new processing chain for real-time GBSAR (RT-GBSAR) is proposed on the basis of the interferometric SAR small baseline subset concept, whereby GBSAR images are processed unit by unit. The outstanding issues have been resolved by the proposed RT-GBSAR chain with three notable features: (i) low requirement of computational memory; (ii) insights into the temporal evolution of surface movements through temporarily-coherent pixels; and (iii) real-time capability of processing a theoretically infinite number of images. The feasibility of the proposed RT-GBSAR chain is demonstrated through its application to both a fast-changing sand dune and a coastal cliff with submillimeter precision

Structural and functional analyses of a sterol carrier protein in Spodoptera litura.

BACKGROUNDS: In insects, cholesterol is one of the membrane components in cells and a precursor of ecdysteroid biosynthesis. Because insects lack two key enzymes, squalene synthase and lanosterol synthase, in the cholesterol biosynthesis pathway, they cannot autonomously synthesize cholesterol de novo from simple compounds and therefore have to obtain sterols from their diet. Sterol carrier protein (SCP) is a cholesterol-binding protein responsible for cholesterol absorption and transport. RESULTS: In this study, a model of the three-dimensional structure of SlSCPx-2 in Spodoptera litura, a destructive polyphagous agricultural pest insect in tropical and subtropical areas, was constructed. Docking of sterol and fatty acid ligands to SlSCPx-2 and ANS fluorescent replacement assay showed that SlSCPx-2 was able to bind with relatively high affinities to cholesterol, stearic acid, linoleic acid, stigmasterol, oleic acid, palmitic acid and arachidonate, implying that SlSCPx may play an important role in absorption and transport of these cholesterol and fatty acids from host plants. Site-directed mutation assay of SlSCPx-2 suggests that amino acid residues F53, W66, F89, F110, I115, T128 and Q131 are critical for the ligand-binding activity of the SlSCPx-2 protein. Virtual ligand screening resulted in identification of several lead compounds which are potential inhibitors of SlSCPx-2. Bioassay for inhibitory effect of five selected compounds showed that AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 inhibited the growth of S. litura larvae. CONCLUSIONS: Compounds AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 selected based on structural modeling showed binding affinity to SlSCPx-2 protein and inhibitory effect on the growth of S. litura larvae

DNA methylation mediates BmDeaf1-regulated tissue- and stage-specific expression of BmCHSA-2b in the silkworm, Bombyx mori

Abstract Background Accurate regulation of tissue- and stage-specific expression of genes is prerequisite for normal development in organisms. DNA methylation plays an important role in modulating gene expression in mammals and plants. However, there is no direct evidence showing how DNA methylation regulates gene transcription in insects. Results During the development of Bombyx mori wing, the expression level of DNA methyltransferase 1 (BmDnmt1) gradually declined and became stationary at pupal stage, resulting in a lower methylation rate of the intragenic promoter of the mid-pupal wing-specific gene BmCHSA-2b, an epidermal chitin synthase controlling mid-pupal wing development in B. mori. The higher methylation rate of the promoter in the pupal epidermis was decreased and BmCHSA-2b transcription was significantly increased by the treatment with the DNA methylation inhibitor, 5-azacytidine-2′-deoxycytidine, suggesting that DNA methylation regulates the tissue-specific expression of BmCHSA-2b. Pupa-specific transcription factor BmDEAF1 bound to the unmethylated intragenic promoter and activated the BmCHSA-2b transcription in the mid-pupal wing. BmDnmt1 and BmDeaf1 influenced the BmCHSA-2b transcription by binding competitively to the CpG island in the promoter. Conclusions All the data together demonstrate that the cooperation between the down-regulation of BmDnmt1 and increased stage-specific expression of BmDeaf1 enhances BmCHSA-2b tissue- and stage-specific transcription to ensure mid-wing development in B. mori. This study highlights an elaborate regulation mechanism how tissue- and stage-specific gene expression is regulated through promoter methylation in insect development

Deep tillage reduces the dependence of tobacco on AMF and promotes the growth of tabacco(Nicotiana tabacum L.) in dryland

The traditional shallow tillage method makes the soil quality declining, and affects the efficiency of agricultural production. Taking the conventional rotary tillage(12 cm) as the control, Yunyan 87 as the test variety, and the soil type of the test site is paddy soil, we studied the effects of deep tillage(Subsoiling 30 cm) on soil nutrients, arbuscular mycorrhizal fungi (AMF) and tobacco（Nicotiana tabacum L.）growth. The results showed that deep tillage increased the contents of organic carbon, available phosphorus(AP) and available potassium(AK) in 20 ~ 40 cm soil layer. The community of AMF was changed by deep tillage. Glomus, the dominant genus in both group, increased significantly in the soil after deep tillage. The colonization rate of AMF was lower than that of conventional rotary tillage. Deep tillage was beneficial to the growth of tobacco in the middle and late stages. Root growth and nutrient content of tobacco increased. Deep tillage significantly improved the output value of tobacco. It can be seen that deep tillage is conducive to improving soil fertility, promoting the vigorous growth of root, reducing the dependence of tobacco on AMF, and promoting the high quality and yield of tobacco in drylands of Hunan.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

A miRNA cluster that contains sli-miR-1814a and sli-miR-3329 in the same EST isolated from <i>S. litura</i>.

<p>A miRNA cluster that contains sli-miR-1814a and sli-miR-3329 in the same EST isolated from <i>S. litura</i>.</p